Compounds for Modulating Integrin CD11B/CD18

ABSTRACT

The application describes an assay for the identification of small molecule modulators of integrin CD11b/CD18 and small molecules capable of modulating activity of this receptor. Such compounds may be used in certain embodiments for treating a disease or condition selected from inflammation, immune-related disorders, cancer, ischemia-reperfusion injury, stroke, neointimal thickening associated with vascular injury, bullous pemphigoid, neonatal obstructive nephropathy, and cardiovascular disease, or in other embodiments for the treatment of a disease or condition selected from immune deficiency, acquired immune deficiency syndrome (AIDS), myeloperoxidase deficiency, Wiskott-Aldrich syndrome, chronic granulomatous disease, hyper-IgM syndromes, leukocyte adhesion deficiency, Chediak-Higashi syndrome, and severe combined immunodeficiency.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under Grant Nos.1R03NS053659 and K01 DK068253 awarded by the National Institutes ofHealth. The United States Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

Integrins are non-covalently linked α/β heterodimeric receptors thatmediate cell adhesion, migration and signaling. Together with theirligands, integrins play central roles in many processes includingdevelopment, hemostasis, inflammation and immunity, and in pathologicconditions such as cancer invasion and cardiovascular disease. The β2integrins, which have a common β-subunit (β2, CD18) but distinctα-subunits (CD11a, CD11b, CD11c and CD11d), are critical leukocytereceptors that are important not only for the function of leukocytes butalso the development of the inflammatory response in vivo. Leukocytesnormally circulate in the vasculature in a quiescent state, but inresponse to inflammatory stimuli, adhere, transmigrate across thevascular endothelium, and enter areas of tissue inflammation where theyparticipate in the destruction and removal of infectious agents and inamplifying the process of inflammation. The integrin CD11b/CD18(complement receptor type 3 (CR3), Mac-1 or αMβ2) is the predominant β2integrin receptor in neutrophils, macrophages and monocytes and mediatesa large number of pro-inflammatory functions in these cells. CD11b/CD18recognizes a wide variety of ligands, including the complement fragmentiC3b, fibrinogen, blood-clotting factor X, CD54 (ICAM-1), the hookwormneutrophil inhibitory factor (NIF), and denatured proteins such asbovine serum albumin (BSA). Studies in CD11b−/− mice have shown thatthis integrin has a distinct and cooperative role (with integrin CD11a)in the inflammatory process. In addition to the knockout mice studies,the biological importance of this integrin in maintaining immunologicalhomeostasis has also been illustrated by different pathologicalconditions where integrins are absent or defective—loss of functional β2integrins causes life-threatening infections in humans and mutationsresult in leukocyte adhesion deficiency type 1, where circulatingneutrophils fail to adhere to or migrate across the endothelium and thepatients are susceptible to recurrent, life-threatening bacterialinfections. Similarly, improper excessive activation of leukocyteintegrins is also harmful, as over-activation of β2 integrinscontributes to sustained inflammation, ischemia-reperfusion injury(including acute renal failure, atherosclerosis and autoimmunedisorders, tissue damage) and the development of various autoimmunediseases. CD11b/CD18 is also implicated in stroke, neointimal thickeningin response to vascular injury2, bullous pemphigoid, and neonatalobstructive nephropathy. Thus, there is a considerable potential foragents that block the binding of CD11b/CD18 to its physiologic ligandsas therapeutics for the treatment of such inflammatory conditions.

Physiologic ligand binding by CD11b/CD is divalent-cation dependent andis mediated by CD11b von Willebrand factor type A (VWFA) domain,CD11bA-domain (A-domain). Blocking anti-CD11b/CD18 antibodies decreasesischemia/reperfusion injury, the area of myocardial infarction and livercell injuries, and diminishes neointimal thickening and restenosis afterballoon injury of carotid arteries in animal models. These antibodiesare also effective in the treatment of endotoxic challenge andhemorrhagic shock and autoimmune injury in various organs including thekidney. However, antibody therapy is not ideal because adverse effectsdue to nonselective blockade of various other leukocyte functions maylead to severe complications. Similarly, neutrophil inhibitory factor(NIF), a 41-kDa glycoprotein ligand-mimic, is effective in attenuatingthe deleterious effects of excessive neutrophil activation in animalmodels, but its large size and immunogenicity preclude its use as atherapeutic agent. Additionally, although blockade of the binding sitesof integrins with ligand-mimetic peptides or small molecules has proveneffective in inhibiting the activities of β1 and β3 integrins, peptidesderived either from CD11b/CD18 ligands or anti-CD11b/CD18 antibodieswere not very efficacious in blocking ligand binding in vitro. Thefailure of these ligand-mimetic peptides to block the interactionbetween iC3b and CD11b/CD18 may be due to their improper conformation insolution or to the size of the ligand binding sites, which may be tooextensive to block with a small peptide.

Current assays for the identification of regulators of CD11b/CD18 relyon purified proteins adsorbed to microtiter plates. Even though theseassays are compatible with high-throughput screening (HTS), purificationof the requisite amount of CD11b/CD18 from mammalian cells for a HTScampaign can be exceedingly difficult and the natural conformation ofintegrin may not be retained upon adsorption to the plastic surfaces.Optimized cell-based phenotypic assays that can be readily utilized inan HTS environment for the rapid identification of small moleculeregulators of this important integrin are currently lacking. Recentreports also suggest that CD11b/CD18 has a central role in theresolution of inflammatory processes by modulating the egress ofadherent neutrophils from the site of inflammation. This suggests thatsmall molecule agonists of CD11b/CD18 may also have a role in treatmentof certain inflammatory and other conditions. Here again, simplecell-based phenotypic assays for ready HTS adaptation are currentlylacking.

Therefore what is needed are small molecules that selectively modulateCD11b/CD18 ligand binding, especially by targeting allosteric regulatorysites, such as the hydrophobic site-for-isoleucine (SILEN) pocket inCD11b/CD1824, which may prove to be a more promising therapeuticstrategy. An effective assay for the rapid identification of smallmolecule modulators of integrin CD11b/CD18 is also needed.

SUMMARY OF THE INVENTION

One aspect of the invention relates to a compound of Formula (I)

-   -   wherein    -   L is absent or is alkyl;    -   Q, W, and Y are independently selected from O and S;    -   X is absent or is selected from O, S, and NR⁴;    -   R¹ is selected from hydrogen, acyl, alkyl, alkenyl, alkynyl,        aryl, heteroaryl, carbocyclyl, heterocyclyl, alkoxycarbonyl,        alkylaminocarbonyl, alkylthiocarbonyl, sulfonate, sulfone,        sulfoxide, and sulfonamide;    -   one of R² and R³ is selected from alkyl, hydroxyalkyl,        aminoalkyl, thioalkyl, alkoxyalkyl, aryl, aralkyl, heteroaryl,        heteroaralkyl, carbocyclyl, carbocyclylalkyl, heterocyclyl, and        heterocyclylalkyl;    -   the other of R² and R³ is selected from hydrogen and alkyl; and    -   R⁴ is selected from hydrogen and alkyl.

One aspect of the invention relates to a compound of Formula (II)

-   -   wherein    -   L is absent or is alkyl;    -   Q and W are independently selected from O and S;    -   X is absent or is selected from O, S, and NR⁵;    -   R¹ is selected from hydrogen, acyl, alkyl, alkenyl, alkynyl,        aryl, heteroaryl, carbocyclyl, heterocyclyl, alkoxycarbonyl,        alkylaminocarbonyl, alkylthiocarbonyl, sulfonate, sulfone,        sulfoxide, and sulfonamide;    -   one of R² and R³ is selected from alkyl, hydroxyalkyl,        aminoalkyl, thioalkyl, alkoxyalkyl, aryl, aralkyl, heteroaryl,        heteroaralkyl, carbocyclyl, carbocycloalkyl, heterocyclyl, and        heterocycloalkyl;    -   the other of R² and R³ is selected from hydrogen and alkyl; and    -   R⁴ and R⁵ are independently selected from hydrogen and alkyl;    -   provided that at least one of R² and R³ is other than hydrogen.

One aspect of the invention relates to a compound of Formula (III)

-   -   wherein    -   A is absent or is selected from C═O, C═S, and SO₂;    -   Q and W are independently selected from O and S;    -   R¹ and R² are independently selected from hydrogen, alkyl,        alkoxy, alkoxyalkyl, hydroxyalkyl, aminoalkyl, thioalkyl,        aralkyl, heteroaralkyl, carbocycloalkyl, and heterocycloalkyl;    -   R³ is selected from hydrogen and alkyl; and    -   R⁴ is selected from alkyl, aralkyl, heteroaralkyl,        carbocycloalkyl, heterocycloalkyl, aryl, heteroaryl,        carbocyclyl, and heterocyclyl.

One aspect of the invention relates to a compound of Formula (IV)

-   -   wherein    -   A and B are independently 5- or 6-membered ring selected from        aryl, carbocycle, heterocycle, and heteroaryl, wherein A and B        together form a fused bicyclic ring system;    -   C is a 5- or 6-membered ring selected from aryl, carbocycle,        heterocycle, and heteroaryl;    -   X is selected from C(R²)(R³), N(R⁴), S, and O;    -   Z is selected from O, S, and NR⁵;    -   R¹ is selected from hydrogen, alkyl, and aralkyl;    -   R² is selected from hydrogen, alkyl, alkoxy, alkoxyalkyl, amino,        aminoalkyl, hydroxy, hydroxyalkyl, thiol, and thioalkyl; or    -   R¹ and R² together are C₁₋₃alkyl or —C₁₋₂alkyl-Z, thereby        forming a 5- to 6-membered ring that is fused to A;    -   R³ is absent or is selected from hydrogen, alkyl, alkoxy,        alkoxyalkyl, amino, aminoalkyl, hydroxy, hydroxyalkyl, thiol,        and thioalkyl; or    -   R⁴ is absent or is selected from hydrogen, alkyl, aryl, aralky,        heteroaryl, heteroaralkyl, carbocyclyl, carbocycloalkyl,        heterocyclyl, and heterocycloalkyl; and    -   R⁵ is selected from hydrogen, alkyl, aryl, aralky, heteroaryl,        heteroaralkyl, carbocyclyl, carbocycloalkyl, heterocyclyl, and        heterocycloalkyl.

In certain embodiments, the invention relates to methods for themodulation of integrin CD11b/CD18 comprising administering a compound ofthe invention.

In certain embodiments, the invention relates to a pharmaceuticalcomposition comprising a compound of the invention and apharmaceutically acceptable diluent or carrier.

In certain embodiments, the invention relates to methods for treating adisease or condition selected from inflammation, immune-relateddisorders, cancer, ischemia-reperfusion injury, stroke, neointimalthickening associated with vascular injury, bullous pemphigoid, neonatalobstructive nephropathy, and cardiovascular disease, comprisingadministering a compound of the invention.

In certain embodiments, the invention relates to an assay for theidentification of small molecule modulators of integrin CD11b/CD18.

In certain embodiments, the invention relates to the use of thedescribed compounds in identification of sites and domains in integrinCD11b/CD18 and in integrin CD11a/CD18 that modulate activity of integrinCD11b/CD18 and in determining exact three-dimensional structure of thebinding pocket, which can be used to derive more selective and/or potentbinders. For example, a complex of CD11b/CD18 with a binding compoundcan be prepared and analyzed, e.g., by x-ray crystallography, nuclearmagnetic resonance, or other suitable means, to identify the bindingsite of CD11b/CD18 that interacts with the compound.

In certain embodiments, computer-based modeling algorithms can be usedto analyze the structures and conformations compounds that bindCD11b/CD18 to identify structural features that contribute to successfulbinding. In certain embodiments, such information is analyzed inconjunction with information about the structure or conformation ofCD11b/CD18 or a binding pocket thereof, such as structural informationobtained by analysis of CD11b/CD18 using analytical techniques such asx-ray crystallography or nuclear magnetic resonance, to analyzeinteractions between binding compounds and the binding pocket theyinteract with. Such analysis can be used to predict the portion ofCD11b/CD18 that interacts with the compound, to select compounds thatpossess structural features correlated with desired binding activityfrom a library of test compounds, or to design structures that areexpected to exhibit binding with CD11b/CD18 for testing in vivo or invitro using assays as described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 a shows surface expression profiling of various stable linesgenerated that express different levels of wild-type CD11b/CD18. FACShistograms showing cells stained with the heterodimer-specific mAb IB4.Mean fluorescence intensity (MFI) for mAb IB4 staining is shown in eachpanel.

FIG. 1 b shows surface expression profiling of one clone (3F9)expressing wild-type CD11b/CD18 at high levels using three differentmAbs. Mean fluorescence intensity (MFI) for mAb staining is shown ineach panel.

FIG. 1 c shows FACS analysis of the reactivity of conformation-sensitivemAb24. MFI values for mAb staining (filled histogram) are shown in eachpanel. No binding was observed with isotype control mAb (non-filledhistogram).

FIG. 1 d shows a bar graph showing the percent input cells (50,000 perwell) adhering to the bottom of fibrinogen-coated wells in the presenceof Ca2+ and Mg2+ (1 mM of each) or of 1 mM Mn2+ and quantitated by themeasurement of cellular ATP levels from a 96-well plate adhesion assayperformed using manual washing of the plate. Each bar represents mean+SDof triplicate determinations from a representative experiment.

FIG. 2 a shows a bar graph showing the number of cells adhering to thebottom of fibrinogen-coated wells in the presence of Ca2+ and Mg2+ (1 mMof each) or of 1 mM Mn2+ and quantitated by measurement of cellular ATPlevels from a 384-well plate adhesion assay performed using automatedwashing of the plate. Each bar represents mean+SD of triplicatedeterminations from a representative experiment. The Z′-values are asindicated.

FIG. 2 b shows photomicrographs from a 384-well plate adhesion assayshowing cells remaining adherent, in the presence of 1 mM Mn2+, before(upper panel) and after (lower panel) automated plate washing.

FIG. 2 c shows photomicrographs from a 384-well plate assay showingcells remaining adherent upon completion of the no-wash cell adhesionassay. Cell adhesion to the uncoated well surface is shown (no block).

FIG. 2 d shows the number of adherent cells as a function of Fg coatingconcentration. Each bar represents mean+SD of triplicate determinationsfrom a representative experiment. Automated microscope coupled withimage analysis software was used to quantify the adherent cells.

FIG. 2 e shows analysis of the assay variability in the no-wash 384-wellassay as measured by quantitation of the adherent cells using automatedmicroscope (left panel), or MTS assay (right panel). Each bar representsmean+SD across 192 wells from a representative experiment.

FIG. 3 shows dose-response curves depicting number of adherent cells inthe presence of increasing amount of blocking mAbs (IB4, left panel;44a, right panel). Concentration of mAb used is indicated at the bottom.Curve fitting was done using XLfit4 to show a dose dependent inhibitionof cell adhesion in the presence of 1 mM Mn2+. Each dot representsmean+SEM of triplicate determinations from a representative experiment.

FIG. 4 shows primary and secondary screening data for selected agonists.Photomicrographs from the primary screen show increased cell adhesioncaused by treatment with three of the identified agonists inphysiological buffer condition. The fold-increase in the number ofadherent cells (over Ca2+ and Mg2+ levels) in the presence of theindicated compounds is shown under each panel. The EC50 value for eachof the compounds obtained from the secondary screen is also indicated.

FIG. 5 shows primary and secondary screening data for selectedantagonists. Bar graphs show percent inhibition of cell adhesion in thepresence of activating Mn2+. Each bar represents mean of duplicatewells. Cell adhesion under physiological condition (TBS++) wasconsidered as maximum inhibition (100%). Cell adhesion observed in thepresence of buffer alone (1 mM Mn2+) was assigned a value of 0% (noinhibition). The IC50 value for each of the compounds obtained from thesecondary screen is also indicated.

FIG. 6 shows dose-response curves depicting number of adherent cells inthe presence of increasing amount of a small molecule. Adhesion of K562CD11b+/CD18+ cells to the integrin CD11b/CD18 ligand fibrinogen or ofK562 CD11a+/CD18+ cells to the integrin CD11a/CD18 ligand ICAM1 wasmeasured in the presence of six different small molecules (IMB8, IMB10,6, 8, 11 and 15). X-axis refers to the concentration of each smallmolecule used and y-axis shows the adherent cell number. Curve fittingwas done using SIGMAPLOT to show a dose dependent increase of celladhesion in the presence of a small molecule. Each dot representsmean+SEM of triplicate determinations from a representative experiment.

FIG. 7 shows that compounds 6 and 11 reduce leukocyte recruitment invivo. Intraperitoneal thioglycolate injection was used to induceperitonitis in mice. PBS buffer alone was used as a control.Approximately five minutes after the thioglycolate injection, the testmice were administered IMB-8, compound 6 or compound 11 intravenously.Cells within the peritoneal cavity were collected after 12 h and countedusing flow cytometry. The data shown are mean (SEM (n=3).

DETAILED DESCRIPTION OF THE INVENTION

Compounds

One aspect of the invention relates to a compound of Formula (I)

-   -   wherein    -   L is absent or is alkyl;    -   Q, W, and Y are independently selected from O and S;    -   X is absent or is selected from O, S, and NR⁴;    -   R¹ is selected from hydrogen, acyl, alkyl, alkenyl, alkynyl,        aryl, heteroaryl, carbocyclyl, heterocyclyl, alkoxycarbonyl,        alkylaminocarbonyl, alkylthiocarbonyl, sulfonate, sulfone,        sulfoxide, and sulfonamide;    -   one of R² and R³ is selected from alkyl, hydroxyalkyl,        aminoalkyl, thioalkyl, alkoxyalkyl, aryl, aralkyl, heteroaryl,        heteroaralkyl, carbocyclyl, carbocyclylalkyl, heterocyclyl, and        heterocyclylalkyl;    -   the other of R² and R³ is selected from hydrogen and alkyl,        preferably R² is hydrogen; and    -   R⁴ is selected from hydrogen and alkyl, preferably hydrogen.

In certain embodiments, Q and W are S and Y is O. In certain otherembodiments, Q and Y are O and W is S.

In certain embodiments, L is alkyl and X is absent or is NR4. In certainsuch embodiments, R1 is selected from aryl, heteroaryl, carbocyclyl,heterocyclyl and alkoxycarbonyl. In certain such embodiments where L isalkyl and X is absent, R1 is selected from carbocyclyl and heterocyclyl,preferably heterocyclyl. In certain such embodiments where L is alkyland X is NR4, R1 is selected from aryl and heteroaryl, preferably aryl.In certain embodiments, where L is alkyl and X is absent, R1 isalkoxycarbonyl.

In certain embodiments, R1 is selected from tetrahydrofuran,tetrahydrothiophene, pyrrolidine, tetrahydropyran, tetrahydrothiopyran,piperidine, piperazine, and morpholine. In certain such embodiments R1is selected from tetrahydrofuran, tetrahydrothiophene, and pyrrolidine,preferably tetrahydrofuran.

In certain embodiments, R1 is phenyl, preferably substituted phenyl. Incertain such embodiments, R1 is phenyl substituted one to five,preferably one to three, more preferably one or two times. In certainsuch embodiments, R1 is phenyl substituted with one or two, preferablytwo substituents independently selected from halogen, nitro, cyano,hydroxyl, thiol, amino, alkoxy, alkylamino, alkylthio, hydroxyalkyl,alkoxyalkyl, aminoalkyl, thioalkyl, and alkyl, more preferably fromalkyl and halogen, e.g., from methyl and chloro.

In certain embodiments, one of R2 and R3 is selected from alkyl,hydroxyalkyl, aminoalkyl, thioalkyl, alkoxyalkyl, aryl, aralkyl,heteroaryl, heteroaralkyl, carbocyclyl, carbocyclylalkyl, heterocyclyl,and heterocyclylalkyl. In certain embodiments, one of R2 and R3 isselected from aryl, aralkyl, heteroaryl, heteroaralkyl, carbocyclyl,carbocyclylalkyl, heterocyclyl, and heterocyclylalkyl, preferably aryl,heteroaryl, carbocyclyl, and heterocyclyl. In certain such embodiments,one of R2 and R3 is selected from aryl and heteroaryl.

In certain embodiments, one of R2 and R3 is heteroaryl selected frompyrrole, furan, and thiophene, preferably furan. In certain embodiments,one of R2 and R3 is furan substituted one to three, preferably one totwo times, more preferably once. In certain such embodiments, one of R2and R3 is furan substituted once with a substituent selected from aryl,aralkyl, heteroaryl, heteroaralkyl, carbocyclyl, carbocyclylalkyl,heterocyclyl, and heterocyclylalkyl, preferably aryl, heteroaryl,carbocyclyl, and heterocyclyl. In certain embodiments, one of R2 and R3is furan substituted once with an aryl group, which itself is optionallysubstituted, preferably one to two times with halogen, e.g.,chlorophenyl or dichlorophenyl.

In certain embodiments, one of R2 and R3 is aryl, preferably phenyl. Incertain such embodiments, one of R2 and R3 is phenyl substituted withone or two, preferably two substituents independently selected fromhalogen, nitro, cyano, hydroxyl, thiol, amino, alkoxy, alkylamino,alkylthio, hydroxyalkyl, alkoxyalkyl, aminoalkyl, thioalkyl, and alkyl.In certain such embodiments, one of R2 and R3 is phenyl substituted oncewith a halogen, preferably bromo.

In certain embodiments, a compound of Formula I is selected from

One aspect of the invention relates to a compound of Formula (II)

-   -   wherein    -   L is absent or is alkyl, preferably absent;    -   Q and W are independently selected from O and S;    -   X is absent or is selected from O, S, and NR⁵, preferably        absent;    -   R¹ is selected from hydrogen, acyl, alkyl, alkenyl, alkynyl,        aryl, heteroaryl, carbocyclyl, heterocyclyl, alkoxycarbonyl,        alkylaminocarbonyl, alkylthiocarbonyl, sulfonate, sulfone,        sulfoxide, and sulfonamide;    -   one of R² and R³ is selected from alkyl, hydroxyalkyl,        aminoalkyl, thioalkyl, alkoxyalkyl, aryl, aralkyl, heteroaryl,        heteroaralkyl, carbocyclyl, carbocycloalkyl, heterocyclyl, and        heterocycloalkyl;    -   the other of R² and R³ is selected from hydrogen and alkyl,        preferably R² is hydrogen; and    -   R⁴ and R⁵ are independently selected from hydrogen and alkyl,        preferably hydrogen.

In certain embodiments, Q is S and W is O.

In certain embodiments, R1 is selected from hydrogen, aryl, heteroaryl,carbocyclyl, and heterocyclyl, preferably aryl or heteroaryl.

In certain embodiments, R1 is heteroaryl selected from pyrazole,imidazole, oxazole, isoxazole, thiazole, and isothiazole, e.g.,thiazole. In certain other embodiments, R1 is hydrogen.

In certain embodiments, R3 is selected from aryl, aralkyl, heteroaryl,heteroaralkyl, carbocyclyl, carbocyclylalkyl, heterocyclyl, andheterocyclylalkyl, preferably aryl, heteroaryl, carbocyclyl,carbocyclylalkyl, and heterocyclyl.

In certain embodiments, R3 is selected from pyrrole, furan, andthiophene, preferably furan. In certain other embodiments, R3 is phenylsubstituted with one or two, preferably one substituent selected fromhalogen, nitro, cyano, hydroxyl, thiol, amino, alkoxy, alkylamino,alkylthio, hydroxyalkyl, alkoxyalkyl, aminoalkyl, thioalkyl, and alkyl.In certain such embodiments, R3 is phenyl substituted with alkoxy, e.g.,wherein the alkoxy group is further substituted with an aryl group,preferably phenyl. In certain such embodiments, R3 is phenyl substitutedwith alkoxy, wherein the alkoxy group is further substituted withhalophenyl, e.g., fluorophenyl.

In certain embodiments, a compound of Formula (II) comprises two aryl orheteroaryl rings, e.g., R3 comprises two aryl or heteroaryl rings, or R3comprises an aryl or heteroaryl ring and R1 comprises an aryl orheteroaryl ring.

In certain embodiments, a compound of Formula (II) is selected from

One aspect of the invention relates to a compound of Formula (III)

-   -   wherein    -   A is absent or is selected from C═O, C═S, and SO₂, preferably        C═O;    -   Q and W are independently selected from O and S;    -   R¹ and R² are independently selected from hydrogen, alkyl,        alkoxy, alkoxyalkyl, hydroxyalkyl, aminoalkyl, thioalkyl,        aralkyl, heteroaralkyl, carbocycloalkyl, and heterocycloalkyl;    -   R³ is selected from hydrogen and alkyl; and    -   R⁴ is selected from alkyl, aralkyl, heteroaralkyl,        carbocycloalkyl, heterocycloalkyl, aryl, heteroaryl,        carbocyclyl, and heterocyclyl.

In certain embodiments, Q is S and W is O.

In certain embodiments, R1 and R2 are independently selected fromhydrogen, alkyl, alkoxy, and alkoxyalkyl, preferably hydrogen and alkyl.In certain such embodiments, R1 and R2 are both hydrogen.

In certain embodiments, R4 is selected from aryl, heteroaryl,carbocyclyl, and heterocyclyl, preferably heteroaryl and heterocyclyl.In certain embodiments, R4 is selected heterocyclyl, preferablycomprising at least two, preferably three fused ring structures. Incertain embodiments, R4 is selected from phenothiazine and phenoxazine,preferably phenothiazine.

In certain embodiments, a compound of Formula (III) is

One aspect of the invention relates to a compound of Formula (IV)

-   -   wherein    -   A and B are independently 5- or 6-membered ring selected from        aryl, carbocycle, heterocycle, and heteroaryl, wherein A and B        together form a fused bicyclic ring system;    -   C is a 5- or 6-membered ring selected from aryl, carbocycle,        heterocycle, and heteroaryl;    -   X is selected from C(R²)(R³), N(R⁴), S, and O, preferably        C(R²)(R³) and N(R⁴);    -   Z is selected from O, S, and NR⁵, preferably O;    -   R¹ is selected from hydrogen, alkyl, and aralkyl;    -   R² is selected from hydrogen, alkyl, alkoxy, alkoxyalkyl, amino,        aminoalkyl, hydroxy, hydroxyalkyl, thiol, and thioalkyl; or    -   R¹ and R² together are C₁₋₃alkyl or —C₁₋₂alkyl-Z, thereby        forming a 5- to 6-membered ring that is fused to A;    -   R³ is absent or is selected from hydrogen, alkyl, alkoxy,        alkoxyalkyl, amino, aminoalkyl, hydroxy, hydroxyalkyl, thiol,        and thioalkyl;    -   R⁴ is absent or is selected from hydrogen, alkyl, aryl, aralkyl,        heteroaryl, heteroaralkyl, carbocyclyl, carbocycloalkyl,        heterocyclyl, and heterocycloalkyl, preferably R⁴ is absent; and    -   R⁵ is selected from hydrogen, alkyl, aryl, aralky, heteroaryl,        heteroaralkyl, carbocyclyl, carbocycloalkyl, heterocyclyl, and        heterocycloalkyl.

In certain embodiments, A is a 5- or 6-membered ring selected from aryland heteroaryl. In certain such embodiments, A is a 6-membered ringselected from aryl and heteroaryl. In certain such embodiments, A isheteroaryl and X is N(R4), wherein R4 is absent. In certain suchembodiments A is selected from pyridine, pyridazine, pyrazine, andpyrimidine, preferably pyrazine. In certain such embodiments, thepyrazine ring is substituted with a halogen, preferably chloro.

In certain such embodiments, B is a 5- or 6-membered ring selected fromaryl and heteroaryl. In certain such embodiments, B is a 5-memberedheteroaryl ring. In certain such embodiments, B is selected frompyrrole, furan, thiophene, pyrazole, imidazole, and furazan, preferablyfurazan. In certain such embodiments R1 is hydrogen.

In certain embodiments, A is 5- or 6-membered ring selected fromcarbocyclyl and heterocyclyl. In certain embodiments, A is a 6-memberedring selected from carbocyclyl and heterocyclyl. In certain suchembodiments, A is a cyclohexa-2,5-dienone.

In certain embodiments, where A is a cyclohexa-2,5-dienone, X isC(R2)(R3). In certain such embodiments, R3 is selected from hydrogen,alkyl, alkoxy, and hydroxy, preferably hydroxy. In certain suchembodiments, R1 and R2 together are C1-3alkyl or —C1-2alkyl-Z, therebyforming a 5- to 6-membered ring that is fused to A, preferably R1 and R2together are —C1-2alkyl-Z, wherein Z is O.

In certain such embodiments, B is a 5- or 6-membered ring selected fromaryl and heteroaryl. In certain such embodiments, B is aryl, preferablyphenyl.

In certain embodiments, C is a 5- or 6-membered ring selected from aryland heteroaryl. In certain such embodiments, C is a 6-membered ring,preferably aryl. In certain such embodiments, C is phenyl or phenylsubstituted with a substituent selected from alkyl, alkoxy, alkylamino,and alkylthio, preferably alkyl. In certain embodiments, C is phenyl ormethylphenyl.

In certain embodiments, a compound of Formula IV is selected from

In certain embodiments, the invention relates to a pharmaceuticalcomposition comprising a compound of the invention and apharmaceutically acceptable diluent or carrier.

In certain embodiments, the invention relates to methods for treating adisease or condition selected from inflammation (including, but notlimited to, inflammatory skin diseases and inflammatory bowel disease),immune-related disorders, autoimmune diseases (such as rheumatoidarthritis), cancer, ischemia-reperfusion injury (including, but notlimited to, acute renal failure, atherosclerosis, autoimmune disorders,and tissue damage), stroke, shock, myocardial infarction, neointimalthickening associated with vascular injury, bullous pemphigoid, neonatalobstructive nephropathy, cardiovascular disease, immune deficiency,acquired immune deficiency syndrome (AIDS), myeloperoxidase deficiency,Wiskott-Aldrich syndrome, chronic granulomatous disease, hyper-IgMsyndromes, leukocyte adhesion deficiency, Chediak-Higashi syndrome, andsevere combined immunodeficiency, comprising administering a compound ofany one of Formulae I to IV. In certain embodiments, the compound isselected from

In certain embodiments, the invention relates to methods for thetreatment of a disease or condition selected from immune deficiency,acquired immune deficiency syndrome (AIDS), myeloperoxidase deficiency,Wiskott-Aldrich syndrome, chronic granulomatous disease, hyper-IgMsyndromes, leukocyte adhesion deficiency, Chediak-Higashi syndrome, andsevere combined immunodeficiency, comprising administering an agonist ofintegrin CD11b/CD18. In certain embodiments, the integrin CD11b/CD18agonist is a compound of any one of Formulae I to III. In certain suchembodiments, the integrin CD11b/CD18 agonist is selected from

In certain embodiments, the invention relates to methods for thetreatment of a disease or condition selected from inflammation(including, but not limited to, inflammatory skin diseases andinflammatory bowel disease), immune-related disorders, autoimmunediseases (such as rheumatoid arthritis), cancer, ischemia-reperfusioninjury (including, but not limited to, acute renal failure,atherosclerosis, autoimmune disorders, and tissue damage), stroke,shock, myocardial infarction, neointimal thickening associated withvascular injury, bullous pemphigoid, neonatal obstructive nephropathy,and cardiovascular disease, comprising administering an antagonist ofintegrin CD11b/CD18. In certain embodiments, the antagonist of integrinCD11b/CD18 is a compound of Formula IV. In certain such embodiments, theantagonist of integrin CD11b/CD18 is selected from

In certain embodiments, the invention relates to methods for themodulation of integrin CD11b/CD18 comprising administering a compound ofthe invention. In certain such embodiments, the compound of theinvention is a compound of any one of Formulae I to IV. In certainembodiments, the invention relates to a method for modulating integrinCD11b/CD18 comprising administering a compound selected from compounds1-43 as designated above.

In certain embodiments, the invention relates to methods for agonizingintegrin CD11b/CD18, comprising administering a compound of theinvention. In certain such embodiments, the compound of the invention isa compound of any one of Formulae I to III. In certain such embodiments,the compound of the invention is selected from

In certain embodiments, the invention relates to methods for inhibitingintegrin CD11b/CD18, comprising administering a compound of theinvention. In certain such embodiments, the compound of the invention isa compound of Formula IV. In certain such embodiments, the compound ofthe invention is selected from

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (d)-isomers, (l)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

If, for instance, a particular enantiomer of a compound of the presentinvention is desired, it may be prepared by asymmetric synthesis, or byderivation with a chiral auxiliary, where the resulting diastereomericmixture is separated and the auxiliary group cleaved to provide the puredesired enantiomers. Alternatively, where the molecule contains a basicfunctional group, such as amino, or an acidic functional group, such ascarboxyl, diastereomeric salts may be formed with an appropriateoptically active acid or base, followed by resolution of thediastereomers thus formed by fractional crystallization orchromatographic means well known in the art, and subsequent recovery ofthe pure enantiomers.

The present invention includes radiolabeled forms of compounds of theinvention, for example, compounds of the invention labeled byincorporation within the structure 3H or 14C or a radioactive halogensuch as 125I.

In certain embodiments, the invention relates to an assay for theidentification of small molecule modulators of integrin CD11b/CD18. Incertain embodiments, the assay is a cell-adhesion-based high-throughputscreening assay. In certain such embodiments, the assay is a no-washcell-adhesion-based high-throughput screening assay, which may, forexample, be performed in a 96- or 384-well plate format. For example,after contacting cells with a solution comprising a test compound on asubstrate treated with a compound that affects (e.g., promotes) celladhesion (such as a ligand for integrin CD11b/CD18), the substrate maybe physically repositioned, e.g., tilted or inverted, such thatnon-adherent cells move away from the substrate by the action ofgravity. In certain embodiments, the samples are treated with afixative, such as formaldehyde. In certain embodiments, the assay can beperformed without contacting the substrate with an additional liquid,e.g., to wash or rinse the substrate. Cells adhering to the substratecan be detected by techniques well known in the art, such as staining orlabel detection or the use of a cell counting reagent, and the resultscompared with a control experiment where a test compound was notincluded.

Alternatively, after contacting cells with a solution comprising a testcompound on a substrate, the solution may be removed from the substrate,e.g., by suction, such as by a robotic apparatus, where, after theremoval of the liquid phase, cells adhering to the substrate can bedetected by techniques well known in the art, such as staining or labeldetection or the use of a cell-counting reagent, and the resultscompared with a control experiment where a test compound was notincluded. In certain embodiments, the samples are treated with afixative, such as formaldehyde, prior to removal of the liquid phasefrom the substrate. In certain embodiments, the assay can be performedwithout contacting the substrate with an additional liquid, e.g., towash or rinse the substrate. While such assays are demonstrated hereinfor identifying agonists or antagonists of integrin C11b/CD18, suchassays can be employed to identify compounds that inhibit or enhancecell adhesion mediated by other mechanisms as well, as will berecognized by those of skill in the art.

DEFINITIONS

The term “alkoxy” refers to an alkyl group having an oxygen attachedthereto. Representative alkoxy groups include methoxy, ethoxy, propoxy,tert-butoxy and the like.

The term “alkoxyalkyl” refers to an alkyl group substituted with analkoxy group and may be represented by the general formulaalkyl-O-alkyl.

The term “alkyl” refers to saturated aliphatic groups, includingstraight-chain alkyl groups, branched-chain alkyl groups, cycloalkyl(alicyclic) groups, alkyl-substituted cycloalkyl groups, andcycloalkyl-substituted alkyl groups. In preferred embodiments, astraight chain or branched chain alkyl has 30 or fewer carbon atoms inits backbone (e.g., C1-30 for straight chains, C3-30 for branchedchains), and more preferably 20 or fewer.

Moreover, the term “alkyl” as used throughout the specification,examples, and claims is intended to include both unsubstituted andsubstituted alkyl groups, the latter of which refers to alkyl moietieshaving substituents replacing a hydrogen on one or more carbons of thehydrocarbon backbone, including haloalkyl groups such as trifluoromethyland 2,2,2-trifluoroethyl, etc.

The term “Cx-y” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups that contain from x to y carbons in the chain. C0alkylindicates a hydrogen where the group is in a terminal position, a bondif internal. A C1-6alkyl group, for example, contains from one to sixcarbon atoms in the chain.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines and salts thereof, e.g., a moietythat can be represented by

-   -   wherein R⁹, R¹⁰, and R^(10′) each independently represent a        hydrogen or a hydrocarbyl group, or R⁹ and R¹⁰ taken together        with the N atom to which they are attached complete a        heterocycle having from 4 to 8 atoms in the ring structure.

The term “aminoalkyl”, as used herein, refers to an alkyl groupsubstituted with an amino group.

The term “aralkyl”, as used herein, refers to an alkyl group substitutedwith an aryl group.

The term “aryl” as used herein include substituted or unsubstitutedsingle-ring aromatic groups in which each atom of the ring is carbon.Preferably the ring is a 5- to 7-membered ring, more preferably a6-membered ring. The term “aryl” also includes polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings is aromatic,e.g., the other cyclic rings can be cycloalkyls, cycloalkenyls,cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls. Aryl groupsinclude benzene, naphthalene, phenanthrene, phenol, aniline, and thelike.

The terms “carbocycle”, “carbocyclyl”, and “carbocyclic”, as usedherein, refers to a non-aromatic saturated or unsaturated ring in whicheach atom of the ring is carbon. Preferably a carbocycle ring containsfrom 3 to 10 atoms, more preferably from 5 to 7 atoms.

The term “carbocycloalkyl”, as used herein, refers to an alkyl groupsubstituted with a carbocycle group.

The term “ether”, as used herein, refers to a hydrocarbyl group linkedthrough an oxygen to another hydrocarbyl group. Accordingly, an ethersubstituent of a hydrocarbyl group may be hydrocarbyl-O—. Ethers may beeither symmetrical or unsymmetrical. Examples of ethers include, but arenot limited to, heterocycle-O-heterocycle and aryl-O-heterocycle. Ethersinclude “alkoxyalkyl” groups, which may be represented by the generalformula alkyl-O-alkyl.

The terms “halo” and “halogen” as used herein means halogen and includeschloro, fluoro, bromo, and iodo.

The term “heteroaralkyl”, as used herein, refers to an alkyl groupsubstituted with a hetaryl group.

The term “heteroaryl” include substituted or unsubstituted aromaticsingle ring structures, preferably 5- to 7-membered rings, morepreferably 5- to 6-membered rings, whose ring structures include atleast one heteroatom, preferably one to four heteroatoms, morepreferably one or two heteroatoms. The term “heteroaryl” also includespolycyclic ring systems having two or more cyclic rings in which two ormore carbons are common to two adjoining rings wherein at least one ofthe rings is heteroaromatic, e.g., the other cyclic rings can becycloalkyls, cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/orheterocyclyls. Heteroaryl groups include, for example, pyrrole, furan,thiophene, imidazole, oxazole, thiazole, pyrazole, pyridine, pyrazine,pyridazine, and pyrimidine, and the like.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, andsulfur.

The term “heterocycloalkyl”, as used herein, refers to an alkyl groupsubstituted with a heterocycle group.

The terms “heterocyclyl”, “heterocycle”, and “heterocyclic” refer tosubstituted or unsubstituted non-aromatic ring structures, preferably 3-to 10-membered rings, more preferably 3- to 7-membered rings, whose ringstructures include at least one heteroatom, preferably one to fourheteroatoms, more preferably one or two heteroatoms. The terms“heterocyclyl” and “heterocyclic” also include polycyclic ring systemshaving two or more cyclic rings in which two or more carbons are commonto two adjoining rings wherein at least one of the rings isheterocyclic, e.g., the other cyclic rings can be cycloalkyls,cycloalkenyls, cycloalkynyls, aryls, heteroaryls, and/or heterocyclyls.Heterocyclyl groups include, for example, piperidine, piperazine,pyrrolidine, morpholine, lactones, lactams, and the like.

The term “hydroxyalkyl”, as used herein, refers to an alkyl groupsubstituted with a hydroxy group.

The term “lower” when used in conjunction with a chemical moiety, suchas, acyl, acyloxy, alkyl, alkenyl, alkynyl, or alkoxy is meant toinclude groups where there are ten or fewer atoms in the substituent,preferably six or fewer. A “lower alkyl”, for example, refers to analkyl group that contains ten or fewer carbon atoms, preferably six orfewer. In certain embodiments, acyl, acyloxy, alkyl, alkenyl, alkynyl,or alkoxy substituents defined herein are respectively lower acyl, loweracyloxy, lower alkyl, lower alkenyl, lower alkynyl, or lower alkoxy,whether they appear alone or in combination with other substituents,such as in the recitations hydroxyalkyl and aralkyl (in which case, forexample, the atoms within the aryl group are not counted when countingthe carbon atoms in the alkyl substituent).

The terms “polycyclyl”, “polycycle”, and “polycyclic” refer to two ormore rings (e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls,heteroaryls, and/or heterocyclyls) in which two or more atoms are commonto two adjoining rings, e.g., the rings are “fused rings”. Each of therings of the polycycle can be substituted or unsubstituted. In certainembodiments, each ring of the polycycle contains from 3 to 10 atoms inthe ring, preferably from 5 to 7.

The term “substituted” refers to moieties having substituents replacinga hydrogen on one or more carbons of the backbone. It will be understoodthat “substitution” or “substituted with” includes the implicit provisothat such substitution is in accordance with permitted valence of thesubstituted atom and the substituent, and that the substitution resultsin a stable compound, e.g., which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, etc.As used herein, the term “substituted” is contemplated to include allpermissible substituents of organic compounds. In a broad aspect, thepermissible substituents include acyclic and cyclic, branched andunbranched, carbocyclic and heterocyclic, aromatic and non-aromaticsubstituents of organic compounds. The permissible substituents can beone or more and the same or different for appropriate organic compounds.For purposes of this invention, the heteroatoms such as nitrogen mayhave hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valences of theheteroatoms. Substituents can include any substituents described herein,for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, analkoxycarbonyl, a formyl, or an acyl), a thiocarbonyl (such as athioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, aphosphate, a phosphonate, a phosphinate, an amino, an amido, an amidine,an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, asulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, aheterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. Itwill be understood by those skilled in the art that the moietiessubstituted on the hydrocarbon chain can themselves be substituted, ifappropriate.

The term “thioalkyl”, as used herein, refers to an alkyl groupsubstituted with a thiol group.

The term “a cell” as used herein includes a plurality of cells.Administering a compound to a cell includes in vivo, ex vivo, and invitro administration.

To “inhibit” or “suppress” or “reduce” a function or activity, such ascancer cell proliferation, is to reduce the function or activity whencompared to otherwise same conditions except for a condition orparameter of interest, or alternatively, as compared to anotherconditions.

The term “modulate” as used herein includes the inhibition orsuppression of a function or activity (such as cell proliferation) aswell as the enhancement of a function or activity.

The phrase “pharmaceutically acceptable” is art-recognized. In certainembodiments, the term includes compositions, excipients, adjuvants,polymers and other materials and/or dosage forms which are, within thescope of sound medical judgment, suitable for use in contact with thetissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically acceptable carrier” as used herein means apharmaceutically acceptable material, composition or vehicle, such as aliquid or solid filter, diluent, excipient, solvent or encapsulatingmaterial useful for formulating a drug for medicinal or therapeutic use.Each carrier must be “acceptable” in the sense of being compatible withother ingredients of the formulation and not injurious to the patient.

Some examples of materials which can serve as pharmaceuticallyacceptable carriers include (1) sugars, such as lactose, glucose andsucrose; (2) starches, such as corn starch and potato starch; (3)cellulose, and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxiccompatible substances employed in pharmaceutical formulations.

The term “pharmaceutically acceptable salt” means an acid addition saltor a basic addition salt which is suitable for or compatible with thetreatment of patients.

The term “pharmaceutically acceptable acid addition salt” as used hereinmeans any non-toxic organic or inorganic salt of any base compoundsrepresented by Formula I or II. Illustrative inorganic acids which formsuitable salts include hydrochloric, hydrobromic, sulfuric andphosphoric acids, as well as metal salts such as sodium monohydrogenorthophosphate and potassium hydrogen sulfate. Illustrative organicacids that form suitable salts include mono-, di-, and tricarboxylicacids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric,fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic,phenylacetic, cinnamic and salicylic acids, as well as sulfonic acidssuch as p-toluene sulfonic and methanesulfonic acids. Either the mono ordi-acid salts can be formed, and such salts may exist in either ahydrated, solvated or substantially anhydrous form. In general, the acidaddition salts of compounds of Formula I or II are more soluble in waterand various hydrophilic organic solvents, and generally demonstratehigher melting points in comparison to their free base forms. Theselection of the appropriate salt will be known to one skilled in theart. Other non-pharmaceutically acceptable salts, e.g. oxalates, may beused, for example, in the isolation of compounds of Formula I or II forlaboratory use, or for subsequent conversion to a pharmaceuticallyacceptable acid addition salt.

The term “pharmaceutically acceptable basic addition salt” as usedherein means any non-toxic organic or inorganic base addition salt ofany acid compounds represented by Formula I or II or any of theirintermediates. Illustrative inorganic bases which form suitable saltsinclude lithium, sodium, potassium, calcium, magnesium, or bariumhydroxide. Illustrative organic bases which form suitable salts includealiphatic, alicyclic, or aromatic organic amines such as methylamine,trimethylamine and picoline or ammonia. The selection of the appropriatesalt will be known to a person skilled in the art.

The term “preventing” is art-recognized, and when used in relation to acondition, such as a local recurrence (e.g., pain), a disease such ascancer, a syndrome complex such as heart failure or any other medicalcondition, is well understood in the art, and includes administration ofa composition which reduces the frequency of, or delays the onset of,symptoms of a medical condition in a subject relative to a subject whichdoes not receive the composition. Thus, prevention of cancer includes,for example, reducing the number of detectable cancerous growths in apopulation of patients receiving a prophylactic treatment relative to anuntreated control population, and/or delaying the appearance ofdetectable cancerous growths in a treated population versus an untreatedcontrol population, e.g., by a statistically and/or clinicallysignificant amount. Prevention of an infection includes, for example,reducing the number of diagnoses of the infection in a treatedpopulation versus an untreated control population, and/or delaying theonset of symptoms of the infection in a treated population versus anuntreated control population. Prevention of pain includes, for example,reducing the magnitude of, or alternatively delaying, pain sensationsexperienced by subjects in a treated population versus an untreatedcontrol population.

The term “solvate” as used herein means a compound of Formula I or II,or a pharmaceutically acceptable salt of a compound of Formula I or II,wherein molecules of a suitable solvent are incorporated in the crystallattice. A suitable solvent is physiologically tolerable at the dosageadministered. Examples of suitable solvents are ethanol, water and thelike. When water is the solvent, the molecule is referred to as a“hydrate”.

As used herein, and as well understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, including clinicalresults. Beneficial or desired clinical results can include, but are notlimited to, alleviation or amelioration of one or more symptoms orconditions, diminishment of extent of disease, stabilized (i.e. notworsening) state of disease, preventing spread of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.

The compositions containing the compounds of the invention can beprepared by known methods for the preparation of pharmaceuticallyacceptable compositions which can be administered to subjects, such thatan effective quantity of the active substance is combined in a mixturewith a pharmaceutically acceptable vehicle. Suitable vehicles aredescribed, for example, in Remington's Pharmaceutical Sciences(Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., USA 1985). On this basis, the compositions include, albeit notexclusively, solutions of the substances in association with one or morepharmaceutically acceptable vehicles or diluents, and contained inbuffered solutions with a suitable pH and iso-osmotic with thephysiological fluids.

The compounds of this invention may be used in the form of the freebase, in the form of salts, solvates and as hydrates. All forms arewithin the scope of the invention. Acid addition salts may be formed andprovide a more convenient form for use; in practice, use of the saltform inherently amounts to use of the base form. The acids which can beused to prepare the acid addition salts include preferably those whichproduce, when combined with the free base, pharmaceutically acceptablesalts, that is, salts whose anions are non-toxic to the animal organismin pharmaceutical doses of the salts, so that the beneficial propertiesinherent in the free base are not vitiated by side effects ascribable tothe anions. Although pharmaceutically acceptable salts of the basiccompounds are preferred, all acid addition salts are useful as sourcesof the free base form even if the particular salt per se is desired onlyas an intermediate product as, for example, when the salt is formed onlyfor the purposes of purification and identification, or when it is usedas an intermediate in preparing a pharmaceutically acceptable salt byion exchange procedures.

Pharmaceutically acceptable salts within the scope of the inventioninclude those derived from the following acids; mineral acids such ashydrochloric acid, sulfuric acid, phosphoric acid and sulfamic acid; andorganic acids such as acetic acid, citric acid, lactic acid, tartaricacid, malonic acid, methanesulfonic acid, ethanesulfonic acid,benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid,quinic acid, and the like.

In accordance with the methods of the invention, the described compoundsor salts or solvates thereof may be administered to a patient in avariety of forms depending on the selected route of administration, aswill be understood by those skilled in the art. The compositions of theinvention may be administered orally or parenterally. Parenteraladministration includes intravenous, intraperitoneal, subcutaneous,intramuscular, transepithelial, nasal, intrapulmonary, intrathecal,rectal and topical modes of administration. Parenteral administrationmay be by continuous infusion over a selected period of time.

A compound of the invention or a salt or solvate thereof may be orallyadministered, for example, with an inert diluent or with an assimilableedible carder, or it may be enclosed in hard or soft shell gelatincapsules, or it may be compressed into tablets, or it may beincorporated directly with the food of the diet. For oral therapeuticadministration, the compound of the invention may be incorporated withexcipient and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.

A compound of the invention may also be administered parenterally orintraperitoneally. Solutions of a compound of the invention as a freebase or pharmacologically acceptable salt or solvate can be prepared inwater suitably mixed with a surfactant such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, DMSO, and mixtures thereof with or without alcohol, and inoils. Under ordinary conditions of storage and use, these preparationscontain a preservative to prevent the growth of microorganisms. A personskilled in the art would know how to prepare suitable formulations.Conventional procedures and ingredients for the selection andpreparation of suitable formulations are described, for example, inRemington's Pharmaceutical Sciences (1990-18th edition) and in TheUnited States Pharmacopeia: The National Formulary (USP 24 NF19)published in 1999.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersion and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringeability exists.

The compounds of the invention may be administered to an animal alone orin combination with pharmaceutically acceptable carriers, as notedabove, the proportion of which is determined by the solubility andchemical nature of the compound, chosen route of administration andstandard pharmaceutical practice.

The dosage of the compounds and/or compositions of the invention canvary depending on many factors such as the pharmacodynamic properties ofthe compound, the mode of administration, the age, health and weight ofthe recipient, the nature and extent of the symptoms, the frequency ofthe treatment and the type of concurrent treatment, if any, and theclearance rate of the compound in the animal to be treated. One of skillin the art can determine the appropriate dosage based on the abovefactors. The compounds of the invention may be administered initially ina suitable dosage that may be adjusted as required, depending on theclinical response.

Exemplification

In Vitro Results

Reagents and Antibodies

Restriction and modification enzymes were obtained from New EnglandBiolabs (Beverly, Mass.), GIBCO BRL (Gaithersburg, Md.) or FisherScientific. Cell culture reagents were from Invitrogen Corp. (San Diego,Calif.) or Fisher Scientific. The anti-CD11b monoclonal antibody (mAb)44a (IgG2a) has been described previously (Arnaout, M. A. et al., J ClinInvest. 1983; 72:171-179) and is available from ATCC. Theheterodimer-specific anti-CD18 mAb IB4 (IgG2a) has also been describedpreviously (Wright, S. D. et al., Proc Natl Acad Sci USA. 1983;80:5699-5703) and is available from ATCC. The ligand mimic mAb 107(IgG1) was generated in-house and has also been previously described(Li, R. et al., J Immunol. 2002; 168:1219-1225) and an activationdependent mAb 24 (IgG1) was previously described (Dransfield, I. andHogg, N., EMBO J. 1989; 8:3759-3765) and is commercially available fromAbcam, MA. Isotype control antibodies MOPC-21 (IgG1) and MOPC-173(IgG2a) and FITC-conjugated goat anti-mouse Ig were obtained fromBDPharmingen (San Diego, Calif.). Human fibrinogen (Plasminogen, vonWillebrand Factor and Fibronectin depleted) was purchased from EnzymeResearch Laboratories (SouthBend, IN). MaxiSorp microtiter plates (96-and 384-well) were from Nunc (Nalgene, NY) and non-fat milk waspurchased from BioRad (Hercules, Calif.).

Cell Culture and Stable Transfection

Stable transfection of wild-type integrin CD11b/CD18 in K562 cells(ATCC) was performed using published protocols (See Hogg, N. et al. JClin Invest. 1999; 103:97-106 and Gupta V. et al. Blood. 2007109:3513-3520).

Briefly, K562 cells (K562 mock) were grown to log phase in Iscove'sModified Dulbecco's Medium (IMDM, CellGro) supplemented with 10%heat-inactivated fetal bovine serum and 50 IU/mL penicillin andstreptomycin, at 37° C. and resuspended in serum-free IMDM at ˜1×107/mL.A total of 0.5 mL of cells were transferred into a 0.4-cm cuvette(Fisher), and 10 g each of linearized wild-type CD11b and wild-type CD18cDNA pcDNA3 expression vectors carrying a G418-resistance marker wasadded. Electroporation was carried out at 960 F and 320V (Gene-Pulser,Bio-Rad). Transfectants were allowed to recover in serum-containingmedia for 48 h and were then selected with 0.5 mg/mL G418 (Invitrogen)for up to two weeks. CD11b/CD18 expressing cells (K562 wild-type) wereenriched by FACS Sorting using the heterodimer specific mAb IB4. Sortedcells were cloned by limiting dilution and clones with varying levels ofintegrin expression were identified by flow cytometry and maintained inIMDM supplemented with 10% heat-inactivated fetal bovine serum, 50 IU/mLpenicillin and streptomycin and 1 mg/mL G418.

Flow Cytometry

Flow cytometric analysis of K562 cells (mock and wild-type) wasperformed using published protocols. Briefly, cells were counted andwashed twice with PBS containing 1 mM each of Ca2+ and Mg2+ ions(PBS++). Cells (5×105) were incubated with primary mAb (3 g/mL) in 100 LPBS++ at room temperature for 15 min, except for mAbs 24 and theisotype-matched control mAb MOPC-21, where incubations were performed inTris Buffered Saline (TBS, Boston Bioproducts) at 37° C. for 30 min andin the presence of either Ca2+ and Mg2+ (1 mM each) or 1 mM Mn2+. Cellswere subsequently washed with PBS++ and incubated with FITC-conjugatedsecondary mAb (5 g/mL) for 20 min at room temperature. Stained cellswere washed and analyzed using FACS Scan flow cytometer (BDBiosciences,CA), counting 10,000 events. Data was analyzed using the CellQuestsoftware (BDBiosciences).

Coating Microtiter Plates with Ligand

Maxisorp microtiter plates (96- and 384-well) were coated with 1-20 g/mLfibrinogen (Fg) ligand in PBS++ overnight at 4° C. Subsequently, theFg-coated wells were washed with TBS and non-specific sites were blockedby incubation with 2% non-fat milk in TBS at room temperature for 30min. Next, the wells were washed three times with TBS and the coatedmicrotiter plates were used in the HTS assays.

HTS Adhesion Assay

All the adhesion assays were carried out in the screening facility atthe Institute of Chemistry and Cell Biology (ICCB), Harvard MedicalSchool using the available small molecule libraries and equipment.Actimol Timtec 1 small molecule library (6,612 compounds) has beenpreviously described(http://iccb.med.harvard.edu/screening/compound_libraries). The smallmolecule library is stocked in 384-well plates at −80° C. and as sealedplates, with each well's containing a unique compound dissolved in DMSOat approximately 5 mg/mL. For the adhesion assay, the K562 cells werewashed with TBS and cells (50,000/well for 96-well plates and20,000/well for 384-well plates) were transferred to the Fg-coated wellsof microtiter plates in the assay buffer (TBS containing 1 mM each ofCa2+ and Mg2+ (TBS++) for agonist identification assays or TBS forantagonist identification assays). Compounds were transferred to eachwell using pin-transfer robot (Seiko D-TRAN XM3106-31 PN 4-axiscartesian robot) with a 384 transfer pin (VP Scientific) calibrated at100 mL and the assay plates were incubated at 37° C. in the presence ofsmall molecule compounds. In the screen for agonists, the cells were inTBS++ and were incubated with small molecules for 30 min. In the screenfor antagonists, following incubation of the cells in TBS with smallmolecules for 20 min at 37° C., 1 mM Mn2+ was added to each well and thecells were further incubated for an additional 15 min at 37° C. In orderto dislodge the non-adherent cell, the assay plates were gently invertedand kept in the inverted position for 20 min at room temperature.Adherent cells were quantitated using either cell viability measuringreagents or automated imaging (see below).

Quantitation of Adherent Cells

For quantitation of adherent cells using a cell viability measuring kit(such as MTS-based CellTiter-One (Promega), CyQuant (Molecular Probes)or Luciferase-based CellTiter-Glo (Promega)), the non-adherent cellswere removed by complete aspiration using an automated liquid handlingmachine (ELx405, Bio-Tek Instruments, VT) and the assay reagent addedaccording to manufacturer's instructions. For automated imaging basedquantitation of adherent cells, the non-adherent cells were fixed byadding a small volume of formaldehyde (1.1% v/v final concentration) andthe plates were kept inverted for an additional 1 h. Upon cell fixation,the wells were washed with TBS using automated liquid handling machine(ELx405) and the adherent cells were fluorescently labeled with DAPI(0.5 M final in TBS with 0.1% TritonX-100) and quantitated usingautomated microscope (see below).

Automated Imaging and Analysis

CellWorx automated microscope (Cellomics, PA) was set at 0.3 s exposureusing DAPI filter set to capture 1-3 images per well. Digitizedphotomicrographs were then analyzed using MetaXpress image analysissoftware (MolecularDevices, CA) using the built-in cell count module toquantify nuclear staining. Data output files were analyzed using MSExcel.

Calculation of Assay Values and Hit Identification

Using the number of non-small molecule treated cells adherent in basalphysiologic buffer condition (1 mM each of Ca2+ and Mg2+) as a minimumthreshold, any small molecule resulting in >10-fold increase in celladhesion was scored as a positive agonist hit. Additionally, using thenumber of non-small molecule treated cells adherent in activating buffercondition (1 mM Mn2+) as a maximum, any small molecule resulting in50%-70% decrease in cell adhesion was scored as a positive antagonisthit.

Statistical Analysis and Curve Fitting

Regression lines were plotted using XLfit4 (ID BusinessSolutions, MA)and EC50 and IC50 values were calculated using GraphPad Prism (SanDiego,Calif.) with four parameter logistic curve fitting using the formula:

y=min+(max−min)/(1+10(log EC50-x)hillslope).

All data are reported as mean+SEM. Z′-factor was calculated as previousdescribed.

Expression of Heterodimeric CD11b/CD18 on K562 Cell Surface

Erythroleukemic K562 cells were chosen for the adhesion assay, as thesecells express integrins, native or recombinant, in a default lowaffinity state similar to normal leukocytes and, as with leukocytes, theexpressed integrins can be activated and made ligand-competent byvarious external stimuli. Thus, these cells provide an excellent contextfor the discovery of small molecule regulators of integrin function. TheK562 cells, which do not endogenously express the integrin CD11b/CD18,were transfected with wild type CD11b/CD18 using electroporation andseveral single cell clones stably expressing the integrin CD11b/CD18 onthe cell surface were obtained by FACS sorting usingheterodimer-specific mAb 1B4 (See McDowall, A. et al., J Clin Invest.2003; 111:51-60), as has been previously described (See Gupta V. et al.Blood. 2007 109:3513-3520 and Annenkov, A. et al., Eur J Immunol. 1996;26:207-212). Several different clones displaying varying levels ofCD11b/CD18 surface expression were also obtained and characterized (FIG.1A), as these clones would be useful in the future for performing doseresponse curves to study the dependence of integrin density on theeffect of various small molecule compounds identified from this screen.One clone, designated 3F9, was selected and the CD11b/CD18 surfaceexpression level on 3F9 was further characterized with anti-CD11b mAb44a and with a ligand mimetic mAb 107, both of which showed bindingcomparable to that observed with the mAb IB4 (FIG. 1B).

The K562 Cell-Surface Expressed Integrin CD11b/CD18 was FunctionallyActive.

K562 cells express wild-type integrin in a largely inactive state, whichcan be activated by inside-out signals primarily through a change inintegrin affinity rather than avidity. The integrin CD11b/CD18expression in the correct conformational state on the surface of K562cells was verified using an activation-sensitive mAb, mAb 24, and usingCD11b/CD18 physiologic ligand fibrinogen. The mAb 24 binds to anactivation- and cation-dependent epitope in the A and has been widelyused as a reporter of the high affinity state in 2 integrins (SeeDransfield, I. and Hogg, N., EMBO J. 1989; 8:3759-3765). The binding ofmAb 24 to CD11b/CD18 expressed on K562 cells was assessed using flowcytometry (FIG. 1C). Very little binding was seen in the low-affinityintegrin conformation in physiologic Ca2+ and Mg2+ (1 mM each) buffer,which reproducibly increased upon activation with 1 mM Mn2+ (FIG. 1C),comparable in magnitude to that observed previously in maximallyactivated 2 integrins expressed in K562 cells, indicating that theintegrins become functionally active in the presence of 1 mM Mn2+.

Adhesion of integrin-expressing cells to ligand-coated plates is a wellknown assay for the study of integrin function and has been used for thestudy of numerous integrins, ligands and cell types. Binding of K562cells to the physiologic CD11b/CD18 ligand fibrinogen (Fg) was analyzedusing 96-well plates. Fg is a symmetric dimer that is recognized by anumber of different integrins and has been shown to be a criticalCD11b/CD18 ligand for inflammatory response and host clearance ofmicrobes. Thus, Fg-CD11b/CD18 interaction is an important target foranti- and pro-inflammatory therapeutic strategies. ELISA-based analysisof Fg-coated wells with anti-Fg mAb showed that the ligand coating ofthe assay plates was highly even and reproducible and displayed very lowvariability. K562 cells (50,000/well) in Tris buffered saline (TBS) wereadded to Fg-coated wells of a 96-well plate, in the presence of EDTA (10mM), Ca2+ and Mg2+ (1 mM each), or Mn2+ (1 mM), and briefly centrifugedat 500 rpm for 10s. After incubation at 37° C. for 30 min, unbound cellswere removed by gentle hand washing and the adherent cells werequantitated using CellTiter-Glo (Promega, Madison, Wis.). In thissystem, no Fg binding by CD11b/CD18-expressing cells was observed in thepresence of EDTA or the physiologic divalent cations Ca2+ and Mg2+ (FIG.1D). Activation with 1 mM Mn2+ induced a large increase in Fg binding bythe CD11b/CD18-expressing cells (FIG. 1D), indicating that the adhesionto Fg was CD11b/CD18 dependent.

Adaptation of Cell Adhesion Assay to a 384-Well Format

Next, the 96-well adhesion assay was directly adapted to the 384-wellplate format. The assay was performed as described for the 96-well plateassay (above), except that fewer K562 cells (20,000/well) were used andthat the final washing step was carried out using an automated platewasher. Although no Fg binding was found by CD11b/CD18-expressing cellsin the absence of activating Mn2+ ions and activation with 1 mM Mn2+induced a large increase in Fg binding by the CD11b/CD18-expressingcells (FIG. 2A), Z′-values ≧0.5 could not be obtained in spite of everyeffort to perform the plate washing as gently as possible. In fact,negative Z′-values were obtained from most of the assays, suggestingthat this protocol presented high variability and was not compatiblewith the HTS environment. Examination of the bottom of 384-well platesfor visualization of 1 mM Mn2+ treated adherent cells usingDAPI-staining and photomicrography with an automated microscope revealedthat uneven number of cells remained adherent upon completion of theassay and sometimes cells were completely absent from the middle of thewells (FIG. 2B). Upon carefully monitoring the adherent cells in thewells after each step in the assay, it was discovered that the automatedwashing step caused substantial and uneven detachment of adherent cellsfrom wells. A gentle inversion of the plates provided a method foreffectively removing non-adherent cells. For the complete removal ofnon-adherent cells prior to quantitation of adherent cells, there weretwo methods developed, both of which gave very similar results.

In the first method, 1.1% formaldehyde was added and the adherent cellswere fixed to the bottom of 384-well plates, in the inverted position,for 1 h at room temperature. The wells were subsequently washed using anautomated plate washer and the fixed cells were stained with DAPI.DAPI-stained cells were photomicrographed using an automated microscopeand the images of stained nuclei were quantitated using MetaXpressfollowing the manufacturer's recommendations. In the second method,using gentle inversion, the supernatant containing non-adherent cellswere completely aspirated using an automated plate washer and adeveloper reagent, such as MTS (which showed the linear dynamicrange >2-logs), was added to quantitate cell number. The plates weredeveloped according to the manufacturer's recommendations.

FIG. 2C shows sample photomicrographs from a 384-well plate showingcells adherent to increasing amount of Fg. These photomicrographs showvery little Fg binding by CD11b/CD18-expressing cells in the absence ofMn2+ and a large increase in binding upon incubation with Mn2+ at everyFg coating concentrations tested. Input cell number was estimated bydetermining cell adhesion to the non-Fg-coated microtiter well surface,which showed high non-specific binding (FIG. 2C, no block). Thephotomicrographs from triplicate measurements were quantitated usingMetaXpress and the results show that the Fg binding byCD11b/CD18-expressing cells was completely dependent upon activatingMn2+ ions and that the variability associated with this assay was verylow (FIG. 2D). Similarly, binding by CD11b/CD18-expressing cells to asecond physiologic ligand iC3b3 showed high selectivity and lowvariability.

Next, the Z′-values across an entire 384-well plate was measured todetermine if the optimized assay was ready for an HTS campaign. FIG. 2Eshows that the Z′-values after completion of the adhesion assay were≧0.5 independent of the method used to quantitate adherent cell number.Although only ˜⅙th of each 384-well was photomicrographed using anautomated microscope (to keep the imaging and image analysis time to aminimum), whereas the entire well was quantitated using MTS, theZ′-values obtained were very similar with the two readouts, suggestingthat this simple protocol presented low variability and highcompatibility with the HTS environment.

The specificity of CD11b/CD18-expressing cells towards Fg in this assayformat was further confirmed by incubation with anti-CD11b (44a) oranti-CD18 (IB4) blocking mAbs. CD11b/CD18-expressing cells werepretreated with an increasing amount of the blocking mAbs for 15 min atroom temperature and then allowed to adhere to Fg-coated wells in thepresence of Mn2+ (1 mM). After incubation at 37° C. for 30 min, unboundcells were removed and the remaining cells were quantitated usingautomated microscopy, which showed a dose-dependent inhibition of celladhesion to Fg (FIG. 3). Both mAbs produced an IC50 value of ˜0.5 g/mL(˜3.3 nM), which is similar to the published value (2 nM) for mAb 44a.Additionally, very little binding was seen with non-transfected K562cells under any condition.

Application of the HTS Adhesion Assay for Identification of SmallMolecule Agonists and Antagonists

The simple, no-wash cell adhesion assay was used to perform a pilotprimary screen to identify small molecule agonists and antagonists usinga library of ˜6,600 compounds (Actimol Timtec1). In the assay foridentification of agonists that produce increased cell-adhesion, thecells were transferred to ligand-coated wells of a 384-well plate in TBSbuffer containing 1 mM each of Ca2+ and Mg2+ (TBS++), and compounds wereadded to each well using a 384 pin-transfer robot. After incubation ofthe assay plate for 30 min at 37° C., the non-adherent cells wereremoved by plate inversion and the cell number in each well wasquantitated using automated microscopy and image analysis. Largeactivation and increase in cell-adhesion (>10 fold increase overadhesion by non-treated cells) was shown for 144 compounds (2.2%). Whilenot wishing to be limited by mechanism, it is believed that compoundsdescribed herein may act directly on the target protein, rather thanthrough other proteins in the cell. FIG. 4 shows data for three of thecompounds that showed high potency in the primary screen (>10-foldincrease in cell-adhesion).

Next, 31 compounds were selected from the initial group of 144 forverification in secondary assays. It was found that 28 of thesecompounds were confirmed agonists (although 4 compounds showed a modest2-fold increase over the background and are thus weak agonists),producing a hit confirmation rate of approximately 90%. The hitconfirmation rate was unusually high for a primary screen and suggeststhat in spite of using a simple, no-wash protocol, the assay had highsensitivity. Additionally, determination of the concentration requiredfor half-maximal increase in cell-adhesion (EC50) for the three selectedagonists showed that even though they displayed similar potency in theprimary screen, Compound 9 was the most potent of all with thecalculated EC50 value of about 6.7 M (FIG. 4), which is in similar rangeas other recently identified agonists.

This assay was also used to identify inhibitors of cell-adhesion(antagonists). Non-adherent cells were removed and the cell number ineach well was quantitated using MTS. Twenty-two compounds (0.3%) showedsignificant (50%-70% decrease) and reproducible (duplicate wells)inhibition. FIG. 5 shows data for three of the compounds that showedhigh potency in the primary screen. The identified compounds contain aplanar aromatic substructure, reminiscent of the small moleculeinhibitors of CD11a A-domain that occupy the allosteric SILEN-pocket(named IDAS in CD11a).

The 22 compounds were then evaluated in secondary assays, wherein nineof the selected compounds (Compounds 22, 23, 24, 25, 27, 31, 36, 38 and41) showed more than 50% inhibition of cell-adhesion and were confirmedantagonists, whereas eight compounds showed no effect. Additionally, thesecondary assays were inconclusive for five compounds, producing a hitconfirmation rate of approximately 53% for antagonists. The threeselected antagonists were further characterized in secondary assays, andall three were highly potent and had very similar IC50 values (FIG. 5).

Selectivity for Integrin CD11b/CD18 Over Integrin CD11a/CD18

A subset of the newly discovered agonists were tested for their activityagainst integrins CD11b/CD18 and CD11a/CD18. The adhesion assays wereperformed in the presence of increasing amounts of each of the selectedsmall molecules and the number of adherent cells under each conditionwas quantitated. The results, presented in FIG. 6, show that expectedlytreatment with control compound IMB-8 showed no increase in celladhesion for either of the two K562 cell lines (K562 cells stablyexpressing integrin CD11b/CD18 or K562 cells stably expressing integrinCD11a/CD18). However, IMB-10 increased equally well the adhesion of bothCD11b/CD18 and CD11a/CD18 expressing K562 cells. Surprisingly, compounds6, 8, 11 and 15 selectively increased adhesion of K562 cells stablyexpressing integrin CD11b/CD18 over K562 cells stably expressingintegrin CD11a/CD18, suggesting that they may be selective agonists ofthe integrin CD11b/CD18 over integrin CD11a/CD18. The calculated EC50value for each of the agonists is also shown in FIG. 6.

In Vivo Results

Coating Microtiter Plates with Ligand

Maxisorp microtiter plates (384-well) were coated with 1-20 g/mLfibrinogen (Fg), 4 g/mL iC3b or 0.375 g/mL ICAM1-Fc ligands in PBS++overnight at 4° C.

Dose Response Curves

The following compounds were selected for measuring dose-responsecurves: 6, 8, 11 and 15. Additionally, IMB-8 and IMB-10 (from Björklund,M. et al., Biochemistry. 2006; 45:2862-2871) were used as controls. Theselected small molecules were purchased from Actimol Timtec (Newark,Del.) and were each dissolved in DMSO at approximately 10 mg/mL and adilution series was created.

For the adhesion assay in 384-well plates, the K562 cells were washedwith TBS and cells (30,000/well) were transferred to the ligand-coatedwells (Fg for CD11b/CD18 and ICAM1-Fc (R&D Systems, Minneapolis, Minn.)for CD11a/CD18) of microtiter plates in the assay buffer (TBS containing1 mM each of Ca2+ and Mg2+ (TBS++). Compounds were transferred to eachwell and the assay plates were incubated at 37° C. in the presence ofsmall molecule compounds and were incubated with small molecules for 30min. In order to dislodge the non-adherent cell, the assay plates weregently inverted and kept in the inverted position for 20 min at roomtemperature. Adherent cells were quantified using either cell viabilitymeasuring reagents or automated imaging (see below).

In Vivo Inflammation Assays

The wild-type (WT) C57BL6 mice of approximately six weeks of age werepurchased from Charles River Labs (Boston, Mass.). Mice were injectedintraperitoneally with 1 mL of 4% w/v sterile thioglycolate (TG) (Sigma,St. Louis, Mich.) in PBS. Control mice were injected with PBS alone.Approximately five minutes after TG injection, 4 g each of compoundsIMB-8, -6 or -11 were administered in 0.5 mL PBS through a tail veininjection. Twelve hours post-injection, the percentage of infiltratedneutrophils in the peritoneal lavage was determined using flowcytometry. Cells from the lavage fluid were labeled with FITC-labeledanti-mouse GR1 and PE-labeled anti-mouse CD11b antibodies (BDPharmingen, Jan Jose, Calif.) and analyzed using FACS Calibur flowcytometer.

Reduction in Acute Inflammatory Response in Thioglycolate-InducedPeritonitis Mouse Model

Novel agonists show marked reduction in neutrophil influx into theperitoneum in a thioglycolate-induced inflammation mouse model of acuteinflammation, as shown in FIG. 7. Injection of non-inflammatory solutionof PBS buffer alone showed almost no neutrophil influx in theperitoneum. However, injection of a solution of 4% thioglycolate in PBSled to a massive influx of neutrophils (bar 2, FIG. 7). Administrationof a control compound (IMB-8), which does not affect function ofintegrin CD11b/CD18 showed no change in the level of neutrophil influxdue to TG injection. However, administration of either compound 6 orcompound II (both integrin CD11b/CD18-selective agonists) showed aremarkable decrease in the number of neutrophils infiltrating to themouse peritoneum due to TG injection.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. An assay for the identification of small molecule modulators of integrin CD11b/CD18, the assay comprising: contacting cells with a solution comprising a test compound on a substrate treated with a compound that affects cell adhesion; physically repositioning the substrate such that non-adherent cells move away from the substrate by the action of gravity; and detecting adherent cells on the substrate.
 2. The assay of claim 1, wherein the cells are K562 cells expressing integrin CD11b/CD18.
 3. The assay of claim 1, wherein the compound that affects cell adhesion promotes cell adhesion to the substrate.
 4. The assay of claim 3, wherein the compound that affects cell adhesion is selected from the group consisting of fibrinogen, iCb3, factor X, ICAM-1, NIF, and denatured BSA.
 5. The assay of claim 1, wherein the solution further comprises Mg²⁺, Ca²⁺, or a mixture thereof.
 6. The assay of claim 1, further comprising removing the solution from the substrate after physically repositioning the substrate.
 7. The assay of claim 6, wherein removing the solution from the substrate comprises aspirating the solution.
 8. The assay of claim 6, wherein removing the solution from the substrate comprises contacting adhered cells with a fixative and washing the substrate.
 9. The assay of claim 8, wherein the fixative comprises formaldehyde.
 10. The assay of claim 1, which is conducted without contacting the substrate with an additional liquid to wash or rinse the substrate.
 11. The assay of claim 1, wherein detecting adherent cells comprises measuring the viability of the adherent cells or imaging the adherent cells.
 12. An assay for the identification of small molecule modulators of an integrin, the assay comprising: coating a substrate with an integrin ligand to form a ligand-coated substrate; contacting the ligand-coated substrate with a solution comprising a test compound and cells expressing the integrin; physically repositioning the substrate to dislodge non-adherent cells; and quantifying adherent cells. 